Toxin-Based Therapeutic Approaches

Shapira, Assaf; Benhar, Itai

Pseudomonas exotoxin A (abbreviated as PE or ETA) is a 613 amino acid polypeptide secreted by the bacterium Pseudomonas aeruginosa as one of its virulence factors [150] . Like diphtheria toxin, it belongs to the family of ADP-ribosylating toxins [151] and to the group of AB toxins (see description of diphtheria toxin above). The toxin can be divided into three main structural and functional domains: The N-terminal domain Ia (aa 1-252) is responsible for cell recognition. Domain II (aa 253-364) is required for the translocation of the toxin across cellular membranes. The exact function of the structural domain Ib (aa is not fully understood. The last four residues (aa 400-404) of domain Ib together with domain III (aa 405-613) form the catalytic subunit of the protein [152, 153] . After the C-terminal Lysine 613 is removed by a plasma carboxypeptidase [154] , leaving the terminus REDL, the toxin binds via its cell-binding domain Ia to CD91, also called alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (α2MR/LRP), on the surface of the cell [155] . The toxin is then internalized and enters early endosomes mainly via clathrin-coated pits, but also via caveosomes, following association with detergent-resistant microdomains [155, 156] . In the acidic environment of the endosome, PE dissociates from its receptor, undergoes a conformational change, and is cleaved by the cellular protease furin in a furin-sensitive loop in domain II of the toxin [123, 157, 158] . Following reduction of the single disulfide bond which holds the proteolytic fragments together [159] , the enzymatic active C' 37 kDa fragment travel in a Rab9-dependent route to the trans-Golgi network (TGN) [160] . There, its C terminal exposed KDEL-like sequence (REDL) binds the KDEL intracellular sorting receptor and the fragment travels to the endoplasmic reticulum (ER) [161] [162] [163] . Alternatively, lipid sorting to the ER may occur [156] . In the ER, sequences in translocation domain II mediates the translocation of the 37 kDa fragment to the cytoplasm in a process that probably involves the subversion of the ER-associated degradation (ERAD) pathway and a retrograde transport via the Sec61p translocon. Escaping, at least in part, from proteosomal degradation may be attributed to the low lysine content of the enzymatically active C terminal 37 kDa fragment [164] [165] [166] [167] [168] [169] . In the cytosol, the APD-ribosylation enzymatic activity of domain III inactivates eEF-2 in a similar way to that of diphtheria toxin (see above), leading to protein synthesis inhibition and programmed cell death [130, 132, [170] [171] [172] [173] (Figure 4 ). domain II and the catalytic (C) domain III (see 3D structure (PDB Entry: 1ikq). In the left panel, the colors of the subunits correspond to those in the scheme. For the sake of simplicity, translocation domain II was extended to contain subdomain Ib). A single disulfide bond bridges between cysteines 265 and 287 within domain II; 2. Following removal of a C terminal lysine residue by plasma carboxipeptidase, the toxin binds to its cell-surface receptor (CD91, also called α2MR/LRP); 3. The toxin is internalized mainly via clathrin-coated pits; 4. In the early endosome (EE), the toxin undergoes conformational change and is cleaved by the protease furin in a furin sensitive loop, in domain II. The two cleavage products remain linked by the intradomain disulfide bond; 5. Following reduction of the disulfid